The present invention relates to a method of detecting biologically active substances affecting intracellular processes, and a DNA construct and a cell for use in the method.
Second messengers and protein kinases play key roles in the signalling pathways that control the response of mammalian cells (and probably all eukaryotic cells) to most stimuli. Although such signalling pathways have been subjected to extensive studies, detailed knowledge on e.g. the exact timing and spatial characteristics of signalling events is often difficult to obtain due to lack of a convenient technology. There is, however, one exception to this rule: our understanding of the role of Ca2+ in e.g. intracellular signalling has been greatly improved due to the development of the fluorescent Ca2+ probe FURA-2 that permits real times studies of Ca2+ in single living cells.
Moreover, the construction of probes of cAMP (Adams et al., Nature 349 (1991), 694-697) and activity of the cAMP-dependent protein kinase (Sala-Newby and Campbell, FEBS 307(2) (1992), 241-244) has been attempted. The protein kinase A probe, however, suffers from the drawback that it is based on the firefly luciferase and accordingly produces too little light for fast single cell mesurements. The cAMP probe on the other hand has to be microinjected and is therefore not well suited for routine laboratoy work.In conclusion, both probes lack some of the elegant properties that resulted in the widespread use of FURA-2.
Recently it was discovered that Green Fluorescent Protein (GFP) expressed in many different cell types, including mammalian cells, becam highly flourescent (Chalfie et al., Science 263 (1994), 802-805). WO/07463 describes a cell capable of expressing GFP and a method for selecting cells expressing a protein of interest and GFP based on detection of GFP-flourescence in the cells.
The purpose of the present invention is to provide a method of detecting a biologically active substance affecting intracellular processes based on the use of green fluorescent protein, including wild-type GFP derived from the jelly fish Aequorea victoria and modifications of GFP, such as modifications that changes the spectral properties of the GFP fluorscence, for the construction of probes, preferably real time probes for second messengers and protein kinase activity.
In one aspect, the present invention relates a DNA construct comprising a DNA sequence coding for
(i) green fluorescent protein (GFP) wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site, or
(ii) a hybrid polypeptide of green fluorscent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site.
In another aspect, the present invention relates to a cell containing a DNA sequence coding for
(i) green fluorscent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition side, or
(ii) a hybrid polypeptide of green fluorescent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site, and capable of expressing said DNA sequence.
In a further aspect, the present invention relates to a method of a detecting a biologically active substance affecting intracellular processes,the method comprising
(a) culturing a cell containing a DNA sequence coding for
(i) green fluorscent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site, or
(ii) a hybrid polypeptide of green flourscent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme reognition site under conditions permitting expression of DNA sequence,
(b) measuring the flourscence of the cell,
(c) incubating the cell with a sample suspected of containing a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorscence measured in step (b), such change being indicative of the prescence of a biologically active substance in said sample.
In a still further aspect, the present invention relates to a method of characterizing the biological activity of a substance with biological activity, the method comprising
(a) culturing a cell containing a DNA sequence coding for
(i) green fluorescent protein wherein one or more amino acids have been substituted, inserted or deleted to provide a binding domain of a second messenger or an enzyme recognition site or
(ii) a hybrid polypeptide of green fluorescent protein (GFP) or a modified GFP and a binding domain of a second messenger or an enzyme recognition site under conditions permitting expression of DNA sequence,
(b) measuring the fluorescence of the cell,
(c) incubating the cell with a sample of a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorscence measured in step (b), said change being characteristic of the biological activity of the biologically active substance in said sample.
Furthermore, studies on the substrate specificity of the different protein kinase A (PKA) isoforms using synthetic peptides have shown that peptides contianing the motifs RRXSX (SEQ ID NO: 33) or RXKRXXSX (SEQ ID NO: 34) (S being the phosphorylated amino acid) tend to be the best substrates for PKA, and a review by Zetterquist, xc3x96, et al. (in Kemp, B. E. (ed). Peptide and Protein Phosphorylation (1990), 172-188, CRC Press, Boca Raton, Fla., U.S.A.) confirms that most known substrates of PKA contain said motifs.
Available amino acid sequences of GFP do not suggest that GFP is a PKA substrate because of a lack of recognition sites comprising the motifs RRXSX (SEQ ID NO: 33) OR RXKRXXSX (SEQ ID NO: 34). It is therefore surprising that a native or wild-type green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria can be phosphorylated by protein kinase A and thereby the spectral properties of GFP are changed resulting in a substantial increase of fluorescence.
In a preferred aspect, the present invention relates to a method of detecting a biologically active substance affecting intracellular processes, the method comprising
(a) culturing a cell containing a DNA sequence coding for a wild-type green fluorescent protein having a protein kinase recognition site under conditions permitting expression of the DNA sequence,
(b) measuring the fluorescence of the cell,
(c) incubating the cell with a sample suspected of containing a biologically active substance affecting intracellular processes, and
(d) measuring the fluorescence produced by the incubated cell and determining any change in the fluorescence compared to the fluorescence measured in step (b), such change being indicative of the presence of a biologically active substance in said sample.
In a further preferred aspect, the present invention relates to a method of characterizing the biological activity of a substance with biological activity, the method comprising
(a) culturing a cell containing a DNA sequence coding for a wild-type green fluorescent protein having a protein kinase recognition site, under conditions permitting expression of the DNA sequence.
(b) measuring the flourescence of the cell,
(c) incubating the cell with a sample of a biologically active substance affecting intracellular processes, and
(d) measuring the flourescence produced by the incubated cell and determining any change in the flourescence compared to the flourescence measured in step (b), said change being characteristic of the biological activity of the biologically active substance in said sample.
In a still further preferred aspect the present invention relates to DNA construct comprising the DNA sequence shown in FIG. 4a, SEQ ID NO: 30, coding for a wild-type GFP an a transformed cell containing said DNA construct and capable of expressing said DNA sequence. The transformed cell is preferably a mammalian cell. A microorganisam Echerichia coli NN049087, carrying the DNA sequence shown in FIG. 4a has been deposited for the prupose of patent procedure according to the Budapest Treaty in the Deutsche Sammlung von Microganismen und Zellkulturen Gmbh, Mascheroderweg 1 b, D-38124 Braunschweig, Germany, under the deposition No. DSM 10260 on Sep. 21, 1995.
In the present context, the term xe2x80x9cgreen fluorescent proteinxe2x80x9d is intended to indicate a protein which, when expressed by a cell, emits fluorscence (cf. Chalfie et al., Science 263, 1994, pp. 802-805). In the following, GFP in which one or more amino acids have been substituted, inserted or deleted is most often termed xe2x80x9cmodified GFPxe2x80x9d.
The term xe2x80x9csecond messengerxe2x80x9d is used to indicate a low molecular weight component invloved in the early events of intracellular signal transduction pathways.
The term xe2x80x9cbinding domain of a second messengerxe2x80x9d is used to indicate a segment of a protein which, in the course of intracellular metabolic proceses, binds the secondary messenger.
The term xe2x80x9cenzyme recognition sitexe2x80x9d is intended to indicate a peptide sequence covalently modified by a enzyme (e.g. phosphorylated, glycosylated or cleaved), preferably the enzyme recognition site is a protein kniase recognition site, which is intended to indicate a peptide sequence covalently modified by a kniase, i.e. phosphorylated.
It should be emphasized that phosphorylation of a protein at a protein kinase recognition site often is followed (or accompanied) by dephosphorylation of said protein. A GFP based probe for activity of given protein kinase(s) would therefore also provide information on the activity of relevant protein phosphatases since the parameter monitored is the net phosphorylation of GFP based probe.
The term xe2x80x9chybrid polypeptidexe2x80x9d is intended to indicate a polypeptide which is a fusion of at least a portion of each of two proteins, in this case at least a portion of the green fluorescent protein and at least a portion of a binding domain of a second messenger or an enzyme recognition site.
In the present context, the term xe2x80x9cbiologically active substancexe2x80x9d is intended to indicate a substance which has a biological function or exerts a biological effect in the human or animal body. The sample may be a sample of a biological material such as a microbial extract, or it may be a sample containing a compound or mixture of compounds prepared by organic synthesis.
The phrase xe2x80x9cany change in fluorescencexe2x80x9d means any change in absorption properties, such as wavelength an intensity, or any change in spectral properties of the emitted light, such as a change of wavelength, fluorescence lifetime, intensity or polarisation.
The mechanism(s) behind a change in e.g. the fluorescence intensity of a modified GFP upon phosphorylation could be several. As one possibility, phosphorylation of said GFP variant could change the chromophore environment, either due to proximity of the added phosphate group or to phosphorylation induced conformation changes. Correspondingly, binding of e.g. a second messenger to the binding domain of a some GFP variant or GFP fusion protein couild induce conformational changes that ultimately changes the chromophore environment and thereby the fluorescence. As support for these suggestions, it has been shown that amino acid substitutions distant to the chromophore (e.g. amino acids 167, 202, 203 and 222) can change the fluorescence intensity and spectral characteristics of GFP (Ehrig et al. (1995) FEBS Letters 367:163; Heim et al.(1994) Proc. Natl. Acad. Sci, 91:12501).
The development of luminescent probes according to the present invention allows real time studies of second messengers and specific enzymes such as protein kinases in single living cells, thereby making it possible to study the precise timing and the spatial characteristics of these factors. Moreover, studies on heterogeneity in cell populations are made possible.
Due to the strong fluorescence of GFP, the luminescence of cells expressing the probes may easily be detected and analyzed by employing a combination of fluorescence microscopy and image analysis. Furthermore, it should be emphasized that the probes described are easy to introduce into cells, as they can be expressed in the cells of interest after transfection with a suitable expression vector.
Real time recombinant probes for second messengers and enzyme activity, such as kinase activity, are not only useful in basic research but also in screening programmes aiming at identifying novel biologically active substances. Many currently used screening programmes designed to find compounds that affect cAMP concentration and protein kinase activity are based on receptor binding and/or reporter gene expression. The recombinant probes described herein, on the other hand, make it possible to develop an entirely new type of screening assays able to monitor immediate and transient changes of cAMP concentration and protein kinase activity in intact living cells.
Any novel feature or combination of features described herein in considered essential to this invention.